Targeting vector to the urokinase plasminogen activator receptor

ABSTRACT

The present invention relates to the targeted delivery of a delivery vehicle construct which specifically binds to and stimulates endocytosis into cells expressing the urokinase plasminogen activator receptor (uPAR), and particularly human airway epithelia. The delivery vehicle construct comprises a portion of uPA and a cargo linked thereto and is useful for the targeted delivery of the cargo to a cell. In one aspect of the invention, the uPA portion of the delivery vehicle construct comprises the wild-type uPA, a fragment of uPA which has the PAI-1 binding region deleted, or a uPA peptide comprising amino acids 13-19 and is useful for the targeted delivery of the cargo to cells, and in particular to airway epithelia. The present invention also provides a method for delivering the delivery vehicle construct to a cell. The method comprises the steps of (a) contacting a target cell with a delivery vehicle construct comprising a uPA portion and a cargo portion; and (b) obtaining a desired result in the target cell.

This application is a continuation of U.S. application Ser. No.10/442,880 filed May 21, 2003, now U.S. Pat. No. 7,132,405 which is acontinuation of U.S. patent application Ser. No. 09/599,846 filed Jun.22, 2000, now U.S. Pat. No. 6,649,597, the contents of which areincorporated herein by reference.

This invention was made with government support under Government NHLBIGrant Number HL 51670. Therefore, the United States Government hascertain rights in the invention.

INTRODUCTION

The present invention relates to the targeted delivery of a deliveryvehicle construct which specifically binds to and stimulates endocytosisinto cells expressing the urokinase plasminogen activator receptor(uPAR), and particularly human airway epithelia. The delivery vehicleconstruct comprises an internalizing portion and a “cargo” portion whichare linked together and is useful for the targeted delivery of the“cargo” to cells expressing uPAR and particularly human airwayepithelia.

BACKGROUND OF INVENTION

Urokinase plasminogen activator (uPA) is expressed in all mammalianspecies. It is produced by many cultured cell types of neoplastic originand has been found more abundantly in explants of tumor tissue than inthe corresponding normal tissue. uPA and its receptor, urokinaseplasminogen activator receptor (uPAR), have been identified in extractsfrom human lung, colon, endometrial, breast, prostate and renalcarcinomas, human melanomas, murine mammary tumors, the murine Lewislung tumor, in ascites from human peritoneal carcinomatosis and humanfibroblasts (Stopelli et al, Proc. Natl. Acad. Sci. USA, 82:4939-43(1985); Vassalli et al., J. Cell. Biol. 100:86-92 (1985), Plow et al.,J. Cell. Biol. 103:2411-2420 (1986), Boyd et al, Cancer Res., 48:3112-6(1988); Nielsen et al., J. Biol. Chem., 263:2358-2363 (1988); Bajpai andBaker, Biochem. Biophys. Res. Commun., 133:994-1000 (1985); Needham etal., Br. J. Cancer, 55:13-16 (1987)).

uPA has been identified as the initiator of a major amplified cascade ofextracellular proteolysis and/or cell migration, presumably through abreakdown of the extracellular matrix, caused by plasmin together withother proteolytic enzymes. This cascade, when regulated, is vital tocertain normal physiological processes but, when dysregulated, isstrongly linked to pathological processes, such as cell invasion andmetastasis in cancer. Dano et al., Adv. Cancer Res. 44:139-266 (1985).There have also been reports that uPA plays a role in (1) thedegradative phase of inflammation, (2) the interference oflymphocyte-mediated cytotoxicity against a variety of cells, (3)angiogenesis, (4) endothelial cell migration which is important in tumorgrowth, and (5) in the cytotoxic effect of natural killer cells.

uPA is a multidomain serine protease comprising (1) an N-terminalepidermal growth factor-like domain, (2) a kringle domain, and (3) aC-terminal serine protease domain. The single chain pro-uPA is activatedby plasmin, which cleaves the chain into the disulfide-linked two chainactive form.

The cellular receptor for uPA is uPAR, which is a multi-domain proteinthat is anchored by a glycolipid to the cell membrane, thus ensuringthat activation of uPA is a pericellular event. Behrendt et al., Biol.Chem., 376:269-79 (1995). uPAR binds the active uPA, as well as pro-uPAand uPA bound to an inhibitor molecule DFP which binds to uPA's activesite. While the receptor binding domain of uPA has been localized toamino acids in the N-terminal growth factor-like domain region, thereare varying studies which have yielded differing results regarding thespan of the receptor binding domain.

For example, Stopelli et al. (Proc. Natl. Acad. Sci. USA, 82:4939-43(1985)) first reported that the N-terminal fragment of uPA (amino acids1-135) was sufficient for high affinity, sub-nanomolar binding to uPAR.Further work confined the uPAR binding domain to amino acids 1-48(Robbiati et al., Fibrinolysis, 4:53-60 (1990)). Dano et al. showed thata region spanning amino acids 12-32 could block the binding of thefull-length wild-type uPA to uPAR (published PCT application WO90/12091). Other studies have shown that residues 20-30 confer thespecificity of binding, but that residues 13-19 are also needed toattain the proper binding confirmation (Appella et al., J. Biol. Chem.,262:4437-40 (1987). Correspondingly, studies have shown that residues20-30 can inhibit the binding of full-length uPA to uPAR but that alonger peptide comprising residues 17-34 is significantly more potent,requiring 10-fold less to achieve the same result. Kobayashi et al., J.Cancer, 57:727-33 (1994). Quax et al. (Arterioscler. Thromb. Vasc.Biol., 18:693-701 (1998) have reported that the receptor binding domainof uPA is localized between amino acids 20-32. In addition, Jones etal., in U.S. Pat. No. 5,942,492 have shown that cyclic peptidescomprising residues 20-30 are sufficient to bind uPAR and act asantagonists of binding of uPA to uPAR and that residues N-terminal toresidue 20 and C-terminal to residue 30 are not essential for highaffinity binding. In view of these studies, the precise location of theuPAR high specificity binding domain in uPA is unclear.

The activity of uPA, when bound to uPAR, is confined to the cell surfaceby plasminogen activator inhibitors (PAI-1 and PAI-2), which bind to andinactivate the uPAR bound uPA. This tight control of uPA activity isnecessary because uPA acts upon a substrate, plasminogen, that ispresent at a high concentration in plasma. Robbins, Meth. Ensemble.,19:184-99 (1970). uPA's action on plasminogen produces plasmin which isa powerful broad spectrum protease that not only degrades extracellularmatrix proteins directly, but also activates the latent forms of otherproteases, including several metalloproteases. Werb et al., N. Eng. J.Med., 296:1017-1023 (1977); Mignatti et al., Cell, 47:487-98 (1986); Heet al., Proc. Natl. Acad. Sci. USA, 86:2632-36 (1989); and Martrisian,Bioessays, 14:455-63 (1992).

In tumor biology, the link between extracellular proteolysis andangiogenesis is clearly evident. Break-up and dissolution of existingextracellular matrix is necessary in order to create new space for bloodvessels to grow into. The processes of proteolysis and angiogenesis arehighly coordinated. For example, two angiogenic growth factors, basicfibroblast growth factor and vascular endothelial growth factor markedlyup-regulate the production of uPA and the expression of uPAR byendothelial cells. Mignatti et al. J. Cell. Biol., 113:1193-1201 (1991);Mandriota et al, J. Biol. Chem., 270:9709-9716 (1995). Therefore, uPAand uPAR have emerged as a target for developing ananti-metastatic/anti-angiogenic therapy for cancer. Fazioli et al,Trends Pharmacological Sci., 15:25-29 (1994).

The uPA/uPAR interaction goes far beyond localizing proteolysis at thecell surface however. The mere occupation of uPAR by uPA induces, byindirect means, signal transduction events leading to one or more of thefollowing effects: mitogenesis (Rabbani et al., J. Biol. Chem.,267:14151-56 (1992)); expression of the c-fos gene (Dumler et al., FEBSLett., 322:37-40 (1994)); cysteine- and metalloprotease expression bymacrophages (Rao et al, J. Clin. Invest., 96:465-74 (1995)); transfer ofmechanical force leading to increased cytoskeletal stiffness (Wang etal., Am. J. Physiol., 268:C1062-66 (1995)); endothelial cell migration(Odekon et al., J. Cellul. Physiol., 150:258-63 (1992)); endothelialcell morphogenesis into tubular structures (Schnaper et al., J. Cellul.Physiol., 165:101-118 (1995)); and endothelial cell deformability andmotility (Lu et al., FEBS Lett. 380:21-24 (1996). All of thesephenomenon are blocked by blocking the access of uPA to uPAR.

In addition to binding uPA, uPAR serves as a cellular adhesion receptorfor vitronectin and as a signaling receptor. Wei et al., J. Biol. Chem.,269:32380-88 (1994); Robinson, Signal transduction via GPI-anchoredmembrane proteins. ADP ribosylation in animal tissue. Plenum Press, NY(1997); Wei et al., J. Cell. Biol., 144:1285-1294 (1999). uPAR alsointeracts with several cell surface proteins including integrins,low-density lipoprotein receptor-related peptide, very-low-densitylipoprotein receptor, megalin and the mannose-6-phosphate/insulin-likegrowth factor-II receptor. Moestrup et al., J. Biol. Chem., 268:16564-70(1993); Heegaard et al, J. Biol. Chem., 270:20855-61 (1995); Czekay etal., Mol. Biol. Cell, 8:517-32 (1997).

uPAR is further involved in both clathrin-dependent andclathrin-independent endocytosis. Vilhardt et al., Mol. Biol. Cell,10:179-195 (1999). Clathrin-dependent endocytosis of uPAR is believed todepend on binding of uPA:PAI-1 to uPAR and subsequent interaction withinternalization receptors for the low-density lipoprotein receptorfamily, which are internalized through clathrin-coated pits. Thisinteraction is inhibited by receptor-associated protein (RAP). Incontrast, clathrin independent endocytosis of uPAR, which is alsobelieved to occur when uPA:PAI-1 is bound, is not inhibited by RAP. SeeVilhardt et al., Mol. Biol. Cell, 10:179-195 (1999).

Rodenberg et al. (Biochem J., 329:55-63 (1998)) have shown thatendocytosis of uPA requires both uPA and PAI-1. The complex of uPA andPAI-1 has been shown to be required for binding to the endocytosisreceptors α2-macroglobulin receptor/low-density lipoproteinreceptor-related protein (α2MR/LRP) and very-low-density lipoproteinreceptor (VLDLR) while free uPA and PAI-1 are not able to bind to theendocytosis receptors. See Rodenberg et al., Biochem J., 329:55-63(1998).

The region of uPA which appears to be responsible for its binding toPAI-1 has been localized to the C-terminal proteinase domain of uPA. Weiet al., J. Biol. Chem., 269:32380-8 (1994); Stoppelli et al., Proc.Natl. Acad. Sci. USA, 82:4939-43 (1985); Conese and Blasi, Biologicaland Chemical Hope-Seyler, 376:143-55 (1995). In addition, Rodenberg etal. have isolated a four residue region of PAI-1 which appears to beresponsible for the high affinity binding of the uPA:PAI-1 complex tothe endocytosis receptors. These references have suggested thatendocytosis of uPA is dependent on PAI-1. Furthermore, the endocytosisof uPA has also been shown to be dependent upon uPAR. Goretzki andMueller, J. Ce. Sci. 110:1395-402 (1997).

The effective treatment of inherited and acquired disorders through thedelivery of a transgene which is capable of correcting the disorder isdependent upon the efficiency of delivery of the transgene. Variousvector systems have been developed that are capable of delivering atransgene to a target cell. However, there remains a need to improve theefficiency of transgene delivery to achieve effective treatments.Improved efficiency is desirable to both increase the ability of thevector to correct the cellular defect and to decrease the toxic effectsof the vector by decreasing the required amount of the vector to achieveeffective treatments.

Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about36 kb. See generally, Horwitz, M. S., “Adenoviridae and TheirReplication,” in Virology, 2nd edition, Fields et al., eds., RavenPress, New York, 1990. Recombinant adenoviruses have advantages for useas delivery systems for nucleic acid molecules coding for, inter alia,proteins, ribozymes, RNAs, antisense RNA that are foreign to theadenovirus carrier (i.e. a transgene), including tropism for bothdividing and non-dividing cells, minimal pathogenic potential, abilityto replicate to high titer for preparation of vector stocks, and thepotential to carry large inserts. See Berkner, K. L., 1992, Curr. Top.Micro Immunol, 158:39-66; Jolly D., 1994, Cancer Gene Therapy, 1:51-64.

Adenoviruses have a natural tropism for respiratory tract cells whichhas made them attractive vectors for use in delivery of genes torespiratory tract cells. For example, adenovirus vectors have been andare being designed for use in the treatment of certain diseases, such ascystic fibrosis (CF): the most common autosomal recessive disease inCaucasians. In CF, mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene disturb cAMP-regulated chloridechannel function, resulting in pulmonary dysfunction. The gene mutationshave been found to encode altered CFTR proteins which cannot betranslocated to the cell membrane for proper functioning. The CFTR genehas been introduced into adenovirus vectors to treat CF in severalanimal models and human patients. Particularly, studies have shown thatadenovirus vectors are fully capable of delivering CFTR to airwayepithelia of CF patients, as well as airway epithelia of cotton rats andprimates. See e.g., Zabner et al., Nature Genetics 6:75-83 (1994); Richet al., Human Gene Therapy 4:461-476 (1993); Zabner et al., Cell75:207-216 (1993); Crystal et al., Nature Genetics 8:42-51 (1994).

However, it would be useful to alter the tropism of a virus, such asadenovirus, to allow it to be used to deliver a nucleic acid molecule toa variety of cells for which the virus is normally non-tropic and toimprove the uptake of the virus into cells.

Certain situations exist where it would be useful to modify the tropismof viruses (such as adenovirus, adeno-associated virus, retrovirus,etc.) to target the vector to cell surface molecules other than thevirus' normal cell surface receptor. For example, certain cells arenormally refractory to infection by certain viruses. It would be usefulto have a method of overcoming the inability of these cells to beinfected. Similarly, in cancer cells, many receptors are up-regulated,such as uPAR. Therefore, it would be useful to be able to specificallytarget vectors to these up-regulated receptors to increase the uptake ofnucleic acids providing for antitumor agents for treatment of suchcancers.

Furthermore, in practice, in order to achieve effective transfer ofviral vectors into affected cells, repeated administration of the viralvector over a course of time may be required. Readministration of theviral vector can trigger an immune response within the subject to whomthe vector is given, which requires subsequently higher doses of theviral vector to avoid immune elimination of the virus. If the efficiencyof uptake of the viral vector is increased, a lower dose of the vectorcan be used, which, in turn, may help alleviate the immune responseproblems which are associated with the readministration of viralvectors. Similar problems may also be alleviated for other types ofvectors as well, such as RNA, polynucleotides, small molecules, etc.

Administration of the cystic fibrosis transmembrane conductanceregulator (CFTR) cDNA to airway epithelia could provide an important newtreatment for cystic fibrosis (CF) lung disease. However, despite thedemonstrated ability of several vectors to deliver and express CFTR andcorrect the Cl⁻ channel defect in airway epithelia in vitro and in vivo(see O'Neal and Beaudet. Hum Mo. Genet. 3:1497-1502 (1994); Crystal.Science 270:404-10 (1995); Wilson. New England J. Med. 334:1185-7(1996); Middleton and Alton. Thorax 53:197-9 (1998); and Welsh. J. Clin.Invest. 105:589-596 (2000), for review)), a severe inefficiency indelivering a vector coding for CFTR has impeded the clinical developmentof the administration of CFTR to CF patients as a new treatment. The twomost important factors for the inefficiency are limited binding ofvector to the apical surface of differentiated human airway epitheliaand limited endocytosis across the apical membrane. A paucity of apicalreceptors prevents binding of viral vectors, including adenovirus(Bergelson et al. Science 275:1320-23 (1997); Pickles et al. J. Virol.72:6014-23 (1998); Walters et al. J. Biol. Chem. 274:10219-10226(1999)), adenovirus-associated virus Teramato et al. J. Virol.72:8904-8912 (1998); Summerford and Samulski. J. Virol. 72:1438-45(1998); Duan et al. Hum. Gene Ther. 9:2761-76 (1998), and retroviralvectors (Wang et al. J. Virol. 72:9818-26 (1998), as well as nonviralvectors, including cationic lipids (Zabner et al. J. Biol. Chem.270:18997-19007 (1995); Matsui et al. J. Biol. Chem. 272:1117-26 (1997);Fasbender et al. Gene Ther. 4:1173-80 (1997)). Compounding the limitedvector binding, and in contrast to observations in cell lines, the rateof endocytosis across the apical membrane of differentiated airwayepithelia is low and may limit administration of adenovirus, AAV,retrovirus and nonviral vectors. Pickles et al. J. Virol. 72:6014-23(1998); Fasbender et al. Gene Ther. 4:1173-80 (1997); and Goldman andWilson. J. Virol. 69:5951-8 (1995).

Hence, a method that increases vector binding and endocytosis mayenhance both viral and non-viral vector administration to airwayepithelia. One strategy to circumvent the lack of apical receptors is tonon-specifically increase vector binding. For example, earlier workshowed enhanced administration of adenovirus or AAV by incorporatingvirus in calcium phosphate (CaPi) coprecipitate. See Fasbender et al. J.Clin. Invest. 102:184-93 (1998); Lee et al. Hum. Gene Ther. 10:603-13(1999); Walters et al. J. Virol. 74:535-540 (2000). However, thisdelivery method did not increase endocytosis across the apical surface.See Walters and Welsh, Gene Ther. 6:1845-1850 (1999); U.S. patentapplication Ser. No. 09/082,510 (incorporated herein by reference).

Other vectors comprising cationic amphiphiles such as lipids, syntheticpolyamino polymers (Goldman et al., 1997, Nat. Biotechnol. 15:462-466),and poly-lysine (Kollen et al., 1996, Hum. Gene. Ther. 7:1577-1586),which are used with nucleic acid molecules, such as a plasmid, totransfect a target cell, are useful for delivery of nucleic acids tocells. Most of these vectors suffer from nonspecificity and inefficiencyof delivery. Therefore, a method for targeting these systems to cellsand improving the uptake of such vectors into cells would also beuseful.

U.S. Pat. No. 5,759,452 ('452 patent) discloses the use of an isolatedportion of the A-chain of a urokinase-type plasminogen activator linkedto a “drug”, wherein the A-chain portion binds stably to an outermembrane of a platelet and delivers the drug to the platelet. Theportion of the A-chain comprises amino acids 1 through 132 or can be thefull-length urokinase-type plasminogen activator. The '452 patentdisclosure is limited to the delivery of a “drug” via the urokinase-typeplasminogen activator in platelets and does not provide teaching for thedelivery to other cell-types, including airway epithelia, and does notprovide teaching for the improved uptake of a vector (including a smallmolecule, protein, polynucleotide, RNA, DNA, virus, viral vector,plasmid, etc) to various cell types.

PCT Patent Application WO98/46632 discloses cyclic uPAR inhibitorpeptides derived from uPA amino acids sequences 22-28 which bind to uPARand block the binding of uPA. WO98/46632 describes the use of thesepeptides for the delivery of diagnostic markers or therapeutic agents touPAR-expressing cells. WO98/46632 states that linear and cyclic peptidescorresponding to amino acids 19-31 of uPA display a surprisingly higheraffinity for uPAR than previously described peptides spanning aminoacids 14-32 of uPA (Magdolen et al., Eur. J. Biochem. 237:743-751 (1996)and 13-19 of uPA (Appella et al., J. Biol. Chem. 262:4437-4440 (1987)which display a low affinity with uPAR. The low affinity of the peptidesdescribed by Magdolen et al. and Appella et al. are described as notbeing sufficient for therapeutic use creating a need for uPA peptideswhich feature higher affinity for the uPAR receptor.

The present invention is based in part on the unexpected finding thatthe seven residue peptide of uPA (corresponding to residues 13-19described by Appella et al. as a low affinity uPAR binder) is capable ofhigh affinity binding and facilitating the delivery and endocytosis ofthe peptide, including the peptide linked to virus (and other cargo) bya cell bearing uPAR, even though this region of uPA does not bind PAI-1which has previously been shown a necessary factor for the endocytosisof uPA. In addition, the present invention is also based on thepreviously unknown finding that airway epithelial cells express uPAR andthat uPA is capable of binding thereto and facilitating endocytosistherein.

These findings have allowed for the targeted and improved delivery ofadenovirus vectors to airway epithelia and is also useful for thetargeted and improved delivery of cargo in general to various cell typesexpressing uPAR.

SUMMARY OF THE INVENTION

The present invention relates to the targeted delivery of a deliveryvehicle construct which specifically binds to and stimulates endocytosisinto cells expressing the urokinase plasminogen activator receptor(uPAR), and in particular, human airway epithelia. The delivery vehicleconstruct comprises a portion of uPA and a cargo linked thereto. Thedelivery vehicle construct is useful for the targeted delivery of cargo,including inter alia, small molecules, polynucleotides (coding, interalia, for a protein, RNA, ribozyme, or antisense RNA), DNA,oligonucleotide decoys, antisense RNA, polypeptides, viruses, modifiedviruses, viral and non-viral vectors and plasmids, to cells expressinguPAR, and in particular, human airway epithelia. Preferred viruses,modified viruses and viral vectors are derived from adenovirus,retrovirus, herpes simplex virus, adeno-associated virus or poxvirus,with adenovirus being particularly preferred.

In one aspect of the invention, the uPA portion of the delivery vehicleconstruct comprises the full-length, wild-type uPA or a fragment of uPAwhich has the PAI-1 binding region deleted and is useful for thetargeted delivery of the cargo to airway epithelia.

In a preferred aspect of the invention, the uPA portion of the vector isa seven residue peptide derived from uPA (u7-peptide) comprising aminoacids 13-19 of uPA (amino acids CLNGGTC; SEQ ID NO:1, where C isCysteine, L is Leucine, N is Asparagine, G is Glycine, and T isThreonine) and the cargo portion is an adenoviral vector, preferablycontaining a transgene. In a particularly preferred embodiment of theinvention, the uPA portion of the vector is the u7-peptide, the cargo isan adenoviral vector containing DNA coding for CFTR and the vector isdelivered to airway epithelia lacking a functional CFTR gene.

The present invention also provides a method for delivering the deliveryvehicle construct to a target cell. The method comprises (a) contactinga target cell with a delivery vehicle construct comprising a uPA portionand a cargo portion; (b) delivering the cargo to the cell; and (c)producing a desired result in the cell. Such desired result may include,inter alia, the correction of a defect in the target cell (e.g.correction of a Cl− channel in the target cell with a transgene encodingCFTR) the destruction of the target cell (e.g. destruction of acancerous cell with a suicide gene), or the delivery of a gene to thetarget cell.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Expression of uPAR in differentiated human airway epithelia. (A)FACS analysis of uPAR staining in COS-1 cells (B) and differentiatedhuman airway epithelia. (C) Projection of 180 μm thick X-Z series ofconfocal images. Green is staining from anti-uPAR antibody and red isethidium bromide fluorescence to identify cells.

FIG. 2: Concentration-dependent binding of uPA to differentiated humanairway epithelia. Data are optical density at 450 nM (O.D.) indicatingcolor change in ELISA assay. n=5.

FIG. 3: uPA stimulation of fluid phase endocytosis by differentiatedhuman airway epithelia measured as uptake of Texas Red dextran. En faceimages (top) and confocal X-Z series (bottom) of labeled dextran uptakeunder basal conditions (A) and following application of 50 nM uPA (B).Dashed line indicates filter. (C) Time-course of dextran uptakefollowing application of uPA (59 nM). (D) Graph shows the effect ofPI-PLC on uPA-mediated stimulation of dextran uptake. n=10. Asteriskindicates p≦0.05.

FIG. 4: Apical fluid phase endocytosis by human airway epitheliastimulated by uPA and u7-peptide (SEQ ID NO:1). Data are en face imagesshowing Texas-Red dextran uptake. Panels are basal endocytosis (A),endocytosis following apical application of 50 nM uPA (B), 0.15 μMu7-peptide (C), or 0.15 μM scrambled u7-peptide (SEQ ID NO:2). (D)Effect of uPA and peptides on a number of cells showing endocytosis.n=5. Asterisk indicates p≦0.05.

FIG. 5: uPA stimulation of gene transfer to normal and CF airwayepithelia. (a) Apical application of uPA (50 nM) prior to infection withAd2/βGal-4 in a CaPi co-precipitate (n=4). (B) Apical application of uPA(50 nM) prior to infection with recombinant AAV in a CaPi co-precipitate(n=10). (C) cAMP-stimulated increase in short-circuit current (ΔIsc) inCF airway epithelia treated with Ad2/CFTR-16 delivered in a CaPicoprecipitate (n=4). Asterisk indicates p≦0.05.

FIG. 6: Endocytosis and gene transfer to freshly excised monkey trachealexplants. (A) Fluid phase endocytosis measured as uptake of FITC-dextranunder basal conditions, or (B) following addition of 50 nM uPA; tracheashows significant background fluorescence. (c) Quantitation ofendocytosis (n=12). (D) X-gal stained explants infected with Ad:CaPicoprecipitates alone or (E) following treatment with 50 nM uPA. (F)Quantitation of gene transfer (n=4). (G) Thin section of X-gal staineduPA treated explant. Asterisks indicate p≦0.05.

FIG. 7: Targeting an adenovirus vector (Ad2/βgal-4) to uPAR by covalentassociation of u7-peptide (SEQ ID NO:1) and PEG to the adenoviruscapsid. “PEG:mutated” refers to a mutated u7-peptide sequence (SEQ IDNO:3) (n=6). Asterisk indicated p≦0.05.

DETAILED DESCRIPTION

The delivery vehicle constructs of the present invention comprise a uPAportion and a cargo portion and can enable the delivery of the cargo(e.g. small molecules; transgenes; polynucleotides, which may code for aprotein, RNA, ribozyme, or antisense RNA; DNA; oligonucleotide decoys;antisense RNA; polypeptides; viruses; modified viruses; viral andnon-viral vectors; and plasmids) to and uptake by cells. The cargo maycomprise a transgene, such as CFTR, which is capable of correcting aninherited or acquired disorder in a subject to which it is administered.

Typically the delivery vehicle carries a transgene which, as definedherein, refers to a nucleic acid molecule not normally present in thevector that codes for, inter alia, a protein, RNA, ribozyme, antisenseRNA. Examples of transgenes include, but are not limited to, CFTR,α-1-antitrypsin AAT, β-glucocerebrosidase, or suicide gene products(suicide gene products may include, inter alia, HSV thymidine kinase(TK), modified TK, cysteine deaminase, E. coli nitroreductase,xanthine-guanine phosphoribosyl transferase, mammalian Pf50 2B1, purinenucleoside, phosphorylase, thymidine phosphorylase, deoxycytidine kinaseand Varicella Zoster virus TK). In a preferred embodiment, the deliveryvehicle construct of the present invention comprises the u7 peptide ofuPA (SEQ ID NO:1) and an adenoviral vector containing a transgene. In aparticularly preferred embodiment, the delivery vehicle construct of thepresent invention comprises the u7 peptide, an adenoviral vectorencoding CFTR and is administered to airway epithelia.

The methods of the present invention enable the delivery vehicleconstruct to be specifically targeted to a cell expressing uPAR on itssurface via the binding of the delivery vehicle construct to uPAR cellsurface molecules. FIG. 1 shows the expression of uPAR in human airwayepithelia. FIG. 2 shows the concentration dependent binding of uPA todifferentiated human airway epithelia and demonstrates that human airwayepithelia, which express uPAR on their cell surface, are capable ofbinding to uPA. It has been discovered by the present inventors that,after binding to the uPAR cell surface molecule of human airwayepithelia, uPA is endocytosed by the cell. See FIG. 3 and FIG. 4. Inaddition, it has been discovered that the delivery vehicle of thepresent invention comprising a uPA portion and a cargo portion alsobinds to and is endocytosed by airway epithelia. See FIG. 5, FIG. 6 andFIG. 7.

The cargo portion of the present invention may comprise any virus orviral vector known to be useful in the art. Examples of such viral cargoinclude: Adenoviridae; Birnaviridae; Bunyaviridae; Caliciviridae,Capillovirus group; Carlavirus group; Carmovirus virus group; GroupCaulimovirus; Closterovirus Group; Commelina yellow mottle virus group;Comovirus virus group; Coronaviridae; PM2 phage group; Corcicoviridae;Group Cryptic virus; group Cryptovirus; Cucumovirus virus group FamilyΦ6 phage group; Cysioviridae; Group Carnation ringspot; Dianthovirusvirus group; Group Broad bean wilt; Fabavirus virus group; Filoviridae;Flaviviridae; Furovirus group; Group Germinivirus; Group Giardiavirus;Hepadnaviridae; Herpesviridae; Hordeivirus virus group; Illarvirus virusgroup; Inoviridae; Iridoviridae; Leviviridae; Lipothrixviridae;Luteovirus group; Marafivirus virus group; Maize chlorotic dwarf virusgroup; icroviridae; Myoviridae; Necrovirus group; Nepovirus virus group;Nodaviridae; Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnipyellow fleck virus group; Partitiviridae; Parvoviridae; Pea enationmosaic virus group; Phycodnaviridae; Picornaviridae; Plasmaviridae;Prodoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxyiridae;Reoviridae; Retroviridae; Rhabdoviridae; Group Rhizidiovirus;Siphoviridae; Sobemovirus group; SSV1-Type Phages; Tectiviridae;Tenuivirus; Tetraviridae; Group Tobamovirus; Group Tobravirus;Togaviridae; Group Tombusvirus; Group Torovirus; Totiviridae; GroupTymovirus; Plant virus satellites.

Additionally, cargo include, but are not limited to, lipid vehicles,poly-lysine vehicles, synthetic polyamino polymer vehicles, and modifiedviral vehicles which are used with nucleic acid molecules, such as aplasmid comprising a transgene.

Particularly preferred cargo are those cargo which have been previouslyemployed for the delivery of transgenes, including, for example,adenovirus, adeno-associated virus, retrovirus, herpes virus andpoxvirus. In a preferred embodiment, the cargo is adenovirus. Normally,adenoviruses bind to a cell surface receptor (CAR) of susceptible cellsvia the knob domain of the fiber protein on the adenovirus surface. Asecondary method of entry for adenovirus is through integrins present onthe cell surface through Arginine-Glycine-Aspartic Acid (RGD) sequencesof the adenoviral penton base protein. The present invention utilizes adelivery vehicle construct which may comprise an adenovirus portion anda uPA portion. The uPA portion of the delivery vehicle construct of thepresent invention allows for improved entry of the adenovirus portion toa target cell.

In another embodiment of the present invention, the cargo of thedelivery vehicle construct is a retrovirus. Retroviruses normally bindto a species specific cell surface receptor (e.g., CD4, CAT, RAM1/GLVR2,GLVR1 and GALV). However, if the cells of interest do not have suchsurface receptors, the virus can not infect such cells. The method ofthe present invention allows the infection of normally refractory cellswith a retrovirus of interest.

Another viral cargo which may be employed in the present invention isadeno-associated virus (AAV). AAV vectors are useful for the delivery oftransgenes to cells because they allow for stable integration oftransgenes into a target cell and they infect a range of cells. Theinherent infection pathway of AAV vectors may be blocked bypre-incubation with neuraminidase. Once the inherent infection pathwayis inhibited, only those cells expressing uPAR on the cell surface willbe infected by the delivery vehicle construct of the present inventioncomprising AAV. This would create a delivery vehicle construct thatwould limit the complication of delivery of a transgene into cells otherthan the target cells. If the objective is to deliver a suicide gene tocancer cells by using the delivery vehicle of the present invention,such a targeted delivery system is particularly desired to avoiddestruction of otherwise healthy cells.

The cargo portion also may include, but is not limited to, smallmolecules, polynucleotides, DNA, oligonucleotide decoys, RNA, antisenseRNA, polypeptides, nonviral vectors and plasmids. Small molecules may betherapeutically useful and may include drugs or other agents which actto ensure proper functioning of a cell or molecules which may induceapoptosis or cell lysis, where death of a cell, such as a cancerouscell, is desired. Nucleic acids may code for, inter alia, a protein,RNA, ribozyme, or antisense RNA. The protein, RNA or ribozyme encoded bythe nucleic acid may allow for the elimination of an undesired functionof a molecule. Decoy oligonucleotides may contain specific binding sitesfor transcription factors and may block the function of thetranscription factors in vitro and in vivo. Where the cargo is apolypeptide, the polypeptide may be a peptide or protein which, whendelivered to the target cell, provides a desired function to the cell orinduces a particular phenotypic alteration.

The uPA portion of the delivery vehicle construct of the presentinvention is “linked” to the cargo portion and facilitates the targetedcellular uptake of the cargo portion. As used herein, the term “link”refers to the connection of the uPA portion to the cargo portion whichmay be any covalent cross-linkage or non-covalent linkage or any othertype of linkage which enables the uPA portion to facilitate delivery anduptake of the cargo portion to cells (e.g. a fusion protein comprisingthe peptide and another protein, the expression of the uPA portion onthe surface of a viral vector, linkage of the uPA portion via chemicalmeans to the cargo portion, etc.) wherein said linkage is between theuPA portion and the cargo portion of the present invention.

One type of linkage comprises a chemical linkage. Any type of chemicallinkage as known in the art can be used, provided the chemicalmodification which creates the linkage does not affect the binding andsubsequent endocytosis of the delivery vehicle to the target cell.

Additional linkers include polymers which may be chemically modified tobe used as linkers. Polymers are large, non-immunogenic, biologicallyinert molecules comprising a chain of smaller molecules linked bycovalent bonds. Polymers useful in accordance with the present inventionare those which when used to covalently or noncovalently link the uPAportion and the cargo portion of the delivery vehicle construct of thepresent invention, provide a polymer-modified delivery vehicle constructthat retains the ability to bind to and be endocytosed by the targetcell. The polymers may be homopolymers or heteropolymers and may belinear or branched. Suitable polymers include polyalkalene compoundssuch as polyalkalene oxides and glycols, e.g. polyethylene glycol (PEG)or polypropylene glycol (PPG).

The uPA portion and the cargo portion may also be linked together by thecreation of a fusion molecule. For example, if the cargo is a protein ora peptide, the uPA portion and the cargo portion may be expressed in arecombinant expression system, such as an E. coli expression system or ayeast expression system, as a fusion comprising the uPA portion and thecargo portion. Additionally, if the cargo is a peptide and the uPAportion is a peptide, the cargo portion and the uPA portion may besynthesized as a continuous peptide. Similar fusion constructs may becreated where the cargo is either DNA or RNA.

As used herein, the uPA portion of the present invention comprises aportion of the urokinase plasminogen activator which is capable ofbinding to and facilitating endocytosis into a target cell. The uPAportion preferably does not include the C-terminal region of uPAresponsible for the binding to PAI-1. In a particularly preferredembodiment, the uPA portion is the u7 peptide (SEQ ID NO:1) and iscapable of binding to the cell surface receptor uPAR and facilitatingendocytosis of the peptide, or any cargo linked thereto, into the targetcell. Where the target cell is any cell expressing uPAR on its cellsurface, the uPA portion is preferably the u7 peptide (SEQ ID NO:1).Where the target cell is airway epithelia, the uPA portion may includeany portion of uPA capable of binding to and facilitating endocytosisinto airway epithelia, including, but not limited to, full lengthwild-type uPA, the u7 peptide (SEQ ID NO:1), and uPA lacking the PAI-1binding portion. Preferably, the uPA is human uPA.

The usefulness of the present invention may be demonstrated, forexample, by incubating the delivery vehicle construct comprising a uPAportion linked to a cargo portion with target cells which express uPARon the cell surface and measuring the efficiency of transfer of theuPA/cargo delivery vehicle construct into the target cell. The deliveryvehicle construct is also useful to identify target cells which aresusceptible to the transfer of the delivery vehicle construct of thepresent invention. Target cells may be identified by incubating thedelivery vehicle construct with various cell types and determining theefficiency of uptake of the delivery vehicle construct into the cell.

In a particular aspect of the invention, it was demonstrated that humanairway epithelia express uPAR by probing human airway epithelia cellswith an anti-uPAR monoclonal antibody and detecting the presence ofbinding of the anti-uPAR monoclonal antibody by fluorescence-activatedcell sorting. See FIG. 1B and Example 1 below. The presence of uPAR onthe apical surface of human airway epithelia was demonstrated usingimmunocytochemistry of unpermeabilized epithelia (FIG. 1C and Example 1below).

It was also demonstrated by a modified ELISA assay that full length uPAbinds to the apical surface of differentiated human airway epithelia.See FIG. 2 and Example 2 below. In one embodiment of the invention, fulllength uPA binds to the apical surface of human airway epithelia at anaffinity of between 0.1 nM to 1.0 mM. In a preferred embodiment of theinvention, full length uPA binds to the apical surface of human airwayepithelia at an affinity of between 20.0 mM to 1.0 μM. In addition, fulllength uPA stimulated increased fluid-phase endocytosis which wasdemonstrated by the uptake of Texas Red-conjugated dextran, a fluidphase marker in the presence of full length uPA. See FIG. 3B and Example3 below. This stimulation of fluid-phase endocytosis was blocked byPI-PLC which cleaves GPI linkages and removes uPAR showing that uPAR isinvolved. (Note, uPAR is a GPI-linked receptor).

As with full length uPA, the u7 peptide of uPA (SEQ ID NO:1), which iskey for binding to uPAR, also stimulated fluid phase endocytosis whereas an equivalent peptide in which the amino acid sequence was scrambled(scrambled peptide; SEQ ID NO:2) did not. See FIG. 4 and Example 3below. The ability of the u7 peptide to stimulate endocytosisdemonstrates that the proteinase domain of uPA and the PAI-1 bindingdomain (previously shown to be required for endocytosis in uPARexpressing cells) are not required for the stimulation of fluid phaseendocytosis.

Furthermore, full length uPA and the u7 peptide enhanced the uptake of anonspecifically bound vector (both an adenoviral vector and an AAVvector). See FIG. 5A and Example 4 below. It has been shown thatincorporating adenovirus or AAV in CaPi precipitates increasesnon-specific binding to the apical surface and thereby enhances genetransfer. See Fasbender et al., J. Clin. Invest. 102:184-193 (1998); Leeet al., Human Gene Ther. 10:603-613 (1999); Walters et al., J. Virol.74:535-540 (2000), all incorporated herein by reference. Theadministration of virus/CaPi coprecipitates does not increase the rateof apical endocytosis however. See Walters and Welsh, Gene Ther.6:1845-1850 (1999) and U.S. patent application Ser. No. 09/082,510, bothincorporated herein by reference. The treatment of airway epithelia witha virus:CaPi complex (serum free) in the presence of uPA and the u-7peptide increased gene delivery 8-10 fold compared to the virus:CaPicomplex alone. See FIG. 5A and Example 4 below. In addition, uPA wasable to enhance gene delivery and could be detected even 2 weeks afterthe virus:CaPi complex was delivered to cells. See FIG. 5B and Example 4below.

The disruption of tight junctions does not account for the increaseduptake of the non specifically bound vector. Full length uPA and the u7peptide also enhanced the uptake of a vector expressing CFTR in cysticfibrosis (CF) airway epithelia. See FIG. 5C and Example 4 below. Theenhanced uptake of the CFTR expressing vector, increased the expressionlevels of CFTR in the airway epithelia. FIG. 5C also shows that acAMP-stimulated current (i.e. corrected the Cl− channel defect in the CFairway epithelia) is created by the increased uptake of theCFTR-expressing vector. In addition, the u7 peptide also corrected theCl− channel defect to a greater extent than the full length uPA.

The ability of uPA to stimulate endocytosis was also demonstrated withfreshly excised monkey tracheal explants. FIG. 6A shows a controlexplant treated with FITC-dextran wherein there was a low level of basalendocytosis. See Example 5 below. The application of uPA stimulated thebasal level endocytosis 3-fold over control levels. See FIG. 6A through6C and Example 5 below. In addition, uPA stimulated uptake of a vectorinto monkey tracheal explants 8-fold over basal levels. See FIGS. 6D, 6Eand 6F and Example 5 below.

To demonstrate that uPA specifically linked to a vector could alsoenhance uptake of the vector into cells, u7-peptide-linked topolyethylene glycol (PEG) was coupled to the capsid of a recombinantadenoviral vector expressing β-galactosidase (βgal) to form au7-PEG-βgal adenovirus complex. Uptake of the u7-PEG-βgal adenoviruscomplex was increased 10-fold as compared to the virus coupled to PEGalone or PEG linked to a mutated peptide (SEQ ID NO:3). See FIG. 7 andExample 6 below.

The present invention further provides physiological compositionsincluding the delivery vehicle constructs comprising a uPA portion and acargo portion of the present invention and further comprising a carrier.Where the physiological compositions are pharmaceutical compositions,they may be prepared by techniques known in the art and reference can bemade to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing,Easton, Pa. The physiological forms of the present compositions suitablefor administration into a subject include sterile aqueous solutions anddispersions. The physiological compositions of the present invention arecompounded for administration in effective dosage amounts with asuitable acceptable carrier and/or diluent.

The precise effective amount of the compositions of the presentinvention can be determined by the ordinary skilled artisan withconsideration of individual differences in age, weight, extent ofdisease and condition of the subject. It can generally be stated thatthe preparation of the present invention should be preferablyadministered in an amount of at least about one plaque forming unit perdesired cell where the cargo is a virus.

It is also advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to discrete units suited as unitarydosages for the subjects to be treated, each unit containing apredetermined quantity of active material calculated to produce thedesired phenotypic effect in association with the required carrier. Thespecification of the novel dosage unit forms of the invention aredictated by and directly depend on the unique characteristics of thecargo and the limitations inherent in the art of compounding. In oneaspect, the cargo of the present invention comprises a viral vectorencoding CFTR, and an effective amount of the composition comprising thecargo is sufficient to correct the Cl⁻ channel defect in a CF subject.

The invention is further illustrated by the following examples which arenot intended in any way to limit the scope of the invention.

EXAMPLES Example 1 Human Airway Epithelia Express uPAR at the ApicalSurface

A. Cells, cultures. COS-1 cells were cultured in Dulbecco's ModifiedEagle's media (DMEM, high glucose) supplemented with 10% fetal bovineserum (FBS; Sigma Chemical Co., St. Louis, Mich.), 100 U/ml penicillinand 100 μg/ml streptomycin (P/S).

Human airway epithelia were obtained from the tracheas and bronchi oflungs removed for organ donation. The University of Iowa In vitro ModelsCore isolated cells by enzyme digestion as described by Yamaya et al.,Am. J. Physiol. 262:L713-724 (1992) and Zabner et al., J. Virol.70:6994-7003 (1996), incorporated herein by reference. Freshly isolatedcells were seeded at a density of 5×10⁵ cells/cm² onto collagen coated,0.6 cm² diameter Millicell polycarbonate filters (Millipore Corp.,Bedford, Mass.). Cultures were maintained in a 37° C. incubator with 7%CO₂ and air. After 24 hours, media from the mucosal side was removed andcells were cultured at the air-liquid interface. Culture media consistedof a 1:1 mixture of DMEM and Ham's F12, 5% Ultraser G (Biosepra SA,Cedex, France), 100 U/ml penicillin, 100 μg/ml streptomycin, 1%nonessential amino acids, and 0.12 U/ml insulin.

B. Fluorescence-activated cell analysis: Human airway epithelia or COS-1cells were washed once with PBS with 0.9 mM Ca²⁺ and 0.9 mM Mg²⁺. Cellswere released from their substratum by a 10 min. incubation in 0.05%trypsin and 0.53 mM EDTA at 37° C. The cell suspension was collected and1 ml of DMEM was added. Cells were centrifuged at 64×g for 5 minutes.Cells were washed with PBS and centrifuged again. Cells were thentreated with either PBS alone or with a murine, anti-human uPAR antibody(1:100, American Diagnostica, Greenwich, Conn.) at 4° C. for 2 hours.Following incubation, cells were centrifuged at 64×g for 5 minutes.Cells were then washed with PBS and incubated with FITC-conjugatedanti-mouse IgG (1:500, Jackson ImmunoResearch, West Grove, Pa.) for 1hour at room temperature. Cells were centrifuged for 5 minutes at 64×g,resuspended in PBS and analyzed by fluorescence-activated cells scans(FACS).

C. Immunocytochemistry: Human airway epithelia were chilled on ice for15 minutes. All incubations were performed at 4° C. and followed by 3washings of PBS. Cells were incubated with 0.05 M glycine for 25 minutesto quench free aldehydes, then washed with 5% BSA for one hour to blocknon-specific binding. Anti-human uPAR antibody (1:100, AmericanDiagnostica) in PBS was added to the apical surface and incubated for 3hours. FITC-conjugated anti-mouse IgG (1:500, Jackson ImmunoResearch) inPBS was added apically for 1 hour. Cells were fixed with 4%paraformaldehyde for 15 minutes, mounted on glass slides, and studiedusing confocal microscopy (MRC-1024, Biorad, Richmond, Calif.). Controlcells were treated with FITC-conjugated anti-mouse IgG alone.

D. Analysis of uPAR Expression on the Apical Surface of Human AirwayEpithelia: To address whether human airway epithelia express uPAR at theapical surface, primary cultures of epithelia were grown at theair-liquid interface under conditions that allow them to differentiateand develop morphological and functional properties of the nativeepithelium. See Yamaya et al., Am J. Physiol. 262:L713-724 (1992);Zabner et al., J. Virol. 70:6994-7003 (1996) (incorporated herein byreference). After at least two weeks in culture, cells were dissociatedfrom the permeable support, probed with an anti-human uPAR monoclonalantibody and labeling was detected by fluorescence-activated cellsorting. FIG. 1A shows a control with COS-1 cells which are known toexpress uPAR. See Nykjaer et al, J. Biol. Chem. 269:25668-76 (1994)(incorporated herein by reference). FIG. 1B demonstrates that humanairway epithelia also express uPAR. In addition, FIG. 1C, which is theimmunocytochemistry of unpermeabilized epithelia, shows that uPAR (greenfluorescence) is expressed on the apical surface of human airwayepithelia. These data are indicative that uPAR may serve as a target forligand applied to the luminal surface of human airway epithelia.

Example 2 uPA Binds uPAR in Airway Epithelia with High Affinity

A. uPA Sources: uPA was purchased from either Calbiochem-Nova BiochemCorp. (human urine, LaJolla, Calif.) or Sigma-Aldrich Inc. (human kidneycells, Milwaukee, Wis.). The u7-peptide (CLNGGTC; SEQ ID NO:1), ascrambled peptide (CTCGNCL; SEQ ID NO:2) and a mutated peptide (CLNFFTC;SEQ ID NO:3) were synthesized by the University of Iowa PeptideSynthesis Core.

B. ELISA on Human Airway Epithelia: Human airway epithelia were chilledon ice for 15 minutes. Following 3 washes in PBS, non-specific bindingwas blocked with 1×TBS (137 mM NaCl, 2.7 mM KCl, 2.5 mM Tris) and 5%milk for 1 hour on ice. All incubations were followed by 3 washes withPBS unless otherwise indicated. Varying concentrations of uPA wereapplied apically for 30 minutes on ice. Cells were then fixed with 4%paraformaldehyde for 15 minutes. Bound uPA was detected by apicalincubation with a mouse monoclonal anti-human uPA antibody (1:1000 in1×TBS+5% milk, American Diagnostica) for 1 hour at 37° C. Anti-mouseIgG-HRP (1:5000 in 1×TBS+5% milk, Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.) was then applied apically for 1 hour at 37° C. TMBDsubstrate buffer (3,3′,5,5′, tetramethyl benzidine dihydrochloride in0.1 M citric acid, 0.2 M Na₂HPO₄, pH 5.0) was added apically for 5minutes at room temperature in the dark. TMBD substrate buffer wasremoved and the reaction was stopped with 2N H₂SO₄. Color change ofsubstrate was read at 450 nm (OD₄₅₀) in a plate reader.

C. Binding of uPA to uPAR in Human Airway Epithelia: Binding of uPA todifferentiated human airway epithelia was tested using the modifiedELISA described above. FIG. 2 shows that uPA bound to the apical surfaceand that half maximal binding occurred at approximately 50 nM.Comparatively, studies in HeLa cells reported an EC50 for binding of uPAto uPAR of approximately 400 nM. See Estreicher et al., J. Biol. Chem.264:1180-9 (1989). The data suggest that a high affinity interactionbetween uPA and uPAR is preserved in human airway epithelia.

Example 3 Apical uPA Increased Fluid-Phase Endocytosis

A. Explants: Freshly excised monkey tracheas were maintained on ice.Explants (1 cm²) were maintained in 1:1 DMEM and Ham's F12 supplementedwith 2% Ultraser G (Biosepra SA) in a 37° C. incubator.

B. Assay for Fluid Phase Endocytosis: Cells were chilled on ice for 15minutes and then incubated with 50 mM uPA or 0.15 μM u7-peptide (SEQ IDNO:1) added to the apical surface for 30 minutes at 4° C. Following 3washes in PBS, cells were incubated with 0.5 mg/ml Texas Red-labeledfixable dextran (3,000 MW, Molecular Probes, Eugene, Oreg.) for 10minutes at 37° C. Following 3 washes in PBS, cells were fixed with 4%paraformaldehyde, mounted onto class slides with Vectashield (VectorLaboratories, Inc., Burlingame, Calif.), and viewed by fluorescencemicroscopy. Control cells were incubated with Texas Red-labeled dextranalone to measure basal levels of endocytosis. Monkey tracheal explantswere assayed as described for primary cell cultures. To evaluatetime-dependent changes, cells were treated as described above with uPA(50 nM) followed by incubations in Texas Red dextran for 5, 10, 30 or 60minutes at 37° C. To evaluate the requirement of GPI-anchored receptorfor uPA stimulated endocytosis, human airway epithelia were incubatedwith phosphatidylinositol-specific phospholipase C prior to incubationswith uPA and Texas Red dextran, (PI-PLC from Bacillus cereus, MolecularProbes) for 2 hours at 37° C. followed by 3 washes in PBS to cleave uPAR(see Hoppe-Seyler, Biol. Chem. 376:143-155 (1995).

B. Effect of uPA on Fluid Phase Endocytosis in Human Airway Epithelia:Stimulation of endocytosis by uPA was determined by measuring the uptakeof Texas Red-conjugated dextran, a fluid phase marker. FIG. 3A showsthat under basal conditions, few human airway epithelia cells took updextran from the apical solution. This result is consistent with earlierwork suggesting a low level of apical endocytosis in airway epithelia.See Pickles et al., J. Virol. 72:6014-23 (1998) and Fasbender et al.,Gene Ther. 4:1173-80 (1997) (incorporated herein by reference).Surprisingly, following apical addition of uPA (50 nM), there was amarked increase in the number of cells that took up the fluid phasemarker (see FIG. 3B). The data suggest that binding of uPA to uPAR inhuman airway epithelia stimulates endocytosis.

C. Determination of Time of Internalization Following uPA Binding: Todetermine how quickly internalization follows ligand binding, humanairway epithelia were incubated with apical uPA (50 nM) at 4° C. for 30minutes. Cells were then incubated with labeled-dextran for 5 to 60minutes at 37° C. An increase in stimulated uptake was apparent by 5minutes and the uptake continued to increase for at least 30 minutes(see FIG. 3C). By 60 minutes, the dextran fluorescence developed a morevesicular appearance, suggesting intracellular accumulation in latestage endosomes or lysosomes. Removal of uPAR by incubation with PI-PLC,which cleaves GPI linkages, eliminated the endocytosis response (seeFIG. 3D). Taken together, the data indicate that uPA potently stimulatesendocytosis in human airway epithelia and that the stimulation is likelymediated through uPAR.

D. Effect of u7-peptide on Fluid Phase Endocytosis in Human AirwayEpithelia: Studies by Appella et al, J. Biol. Chem. 262:4437-40 (1987)(incorporated herein by reference) identified a N-terminal portion ofuPA that is key for binding to uPAR comprising residues 13-19 (CLNGGTC;SEQ ID NO:1). The U7-peptide was synthesized and examined for itsability to stimulate endocytosis. As with full length uPA, applicationof the u7-peptide (SEQ ID NO:1) to the apical surface of human airwayepithelia stimulated fluid phase endocytosis, whereas an equivalentpeptide in which the sequence was scrambled (scrambled peptide; SEQ IDNO:2) did not (see FIG. 4). The data show that binding of a smallpeptide from uPA which does not comprise the C-terminus of uPA, nor theproteinase domain of uPA containing the PAI-1 binding region of uPA,stimulated endocytosis. Since the u7-peptide does not contain theproteinase domain containing the sequences previously shown to berequired for uPA to bind PAI-1, these data also show that uPA:PAI-1complex surprisingly is not required to stimulate endocytosis in humanairway epithelia.

Example 4 uPA Enhances Gene Transfer by Non-Specifically Bound Vector toHuman Airway Epithelia

A. Recombinant Adenovirus and AAV: Recombinant adenovirus vectorsexpressing β-galactosidase (Ad2/β-Gall-4; U.S. Pat. No. 5,882,877,incorporated herein by reference) and CFTR (Ad2/CFTR-16; PCT PublicationWO/9846781, incorporated herein by reference) were prepared as describedpreviously by Lee et al., Human Gene Ther. 10:603-13 (1999)(incorporated herein by reference) by the University of Iowa GeneTransfer core at titers of approximately 10¹⁰ infectious units/ml.Recombinant AAV2 vector expressing β-galactosidase was provided by Cf.Jay Chiorini (NIH) at approximately 1×10⁷ IU/ml (Chiorini et al., J.Virol. 73:1309-1319 (1999)).

B. Evaluation of Gene Transfer: The apical surface of human airwayepithelia was washed once with PBS. Adenovirus was applied at amultiplicity of infection (MOI) of 50 to the apical surface for 30minutes at 37° C. Adenovirus was applied either alone or in a CaPicoprecipitate as previously described by Fasbender et al., J. Clin.Invest. 102:184-193 (1998) and Lee et al., Hum. Gene Ther. 10:603-13(1999) (incorporated herein by reference). Following 3 washes, cellswere returned to the incubator and assayed for gene transfer 48 hourslater. AAV2 was applied at an MOI<1 to the apical surface for 30 minutesat 37° C. AAV2 was applied either alone or on a CaPi coprecipitateformed as previously described by Fasbender et al. and Lee et al. foradenovirus. Following 3 washes, cells were returned to the incubator andassayed for gene transfer 14 days later. Total β-galactosidase activitywas measured using a commercially available technique (Galacto-Light,Topix, Bedford, Mass.).

Monkey tracheal explants were infected as described for primary cells.Following infection, explants were maintained in a 37° C. incubator for72 hours and then stained with X-gal. For X-gal staining, explants werefixed with 1.8% formaldehyde and 2% glutaraldehyde, and then incubatedat 37° C. in X-gal solution overnight.

C. Measurement of Transepithelial Electrical Properties: Epithelia weremounted in modified Ussing chambers (Jim's Instruments, Iowa City, Iowa)and bathed with Ringer's solution on their submucosal side (135 mM NaCl,2.4 K₂HPO₄, 0.6 mM KH₂PO₂, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 5 mM Hepes, pH7.4 and 10 mM dextrose). The mucosal surface was bathed in the identicalsolution, except that 135 mM NaGluconate replaced the 135 mM NaCl. Inthis way, a transepithelial Cl⁻ concentration gradient was establishedin order to magnify changes in Cl⁻ transport. The transepithelial Na⁺transport was blocked with mucosal amiloride (10 μM). Mucosal DIDS (100μM) blocked non-CFTR Cl⁻ transport. The cAMP agonists, forskolin (10 μM)and IBMX (100 μM) were applied to both solutions to stimulatetransepithelial Cl⁻ transport via CFTR.

D. uPA Enhancement of Gene Transfer: The ability of uPA to enhance genetransfer by nonspecifically bound vector was tested. Incorporation ofadenovirus or AAV in CaPi coprecipitates increases non-specific virusbinding to the apical surface and thereby enhances gene transfer.However, administration of CaPi coprecipitates, alone, does not increasethe rate of apical endocytosis and depends upon the basal rate ofendocytosis. In contrast, the addition of uPA (50 nM) to the apicalsurface of human airway epithelia and the subsequent administration ofCaPi:adenovirus coprecipitates increased gene transfer 8-10 fold ascompared to the coprecipitate alone when assayed for gene transferforty-eight hours after the administration of the coprecipitates (seeFIG. 5A). Similar results were also obtained with CaPi:AAVcoprecipitates; FIG. 5B shows that uPA enhanced gene transfer whenmeasured 2 weeks after AAV was delivered in a CaPi coprecipitate. Noeffect was seen on the integrity of tight junctions which has previouslybeen shown to allow access of viral vectors to the basolateral surfacewhere they bind receptors. The u7-peptide (SEQ ID NO:1), which has noenzymatic activity, had an effect similar to uPA, and stimulated fluidphase endocytosis across the apical membrane. Finally, disrupting tightjunctions by chelating Ca²⁺ did not enhance gene transfer byAd/βgal:CaPi. Tight junction integrity was measured by transepithelialresistance which did not change following uPA or u7 treatment. Thesedata indicate that disruption of tight junctions did not account for theenhanced gene transfer.

The ability of uPA to enhance gene transfer and expression of CFTR indifferentiated CF airway epithelia was also tested. FIG. 5C shows thatgene transfer with an adenovirus expressing CFTR in a CaPi coprecipitategenerated a cAMP-stimulated current. The u7-peptide further enhanced thecorrection. The data show that the u7-peptide targets cells involved ingenerating transepithelial Cl⁻ transport.

Example 5 The Ability of uPA to Stimulate Endocytosis and Gene Transferin Monkey Tracheal Explants

As an additional test of uPA-mediated endocytosis and gene transfer,freshly excised monkey tracheal explants were tested. FIG. 6A shows acontrol explant treated with FITC-dextran only, as with human epithelia,there was a low level of basal endocytosis. In contrast, apicalapplication of uPA stimulated endocytosis 3-fold over control levels(see FIG. 6A through 6C). The effect of uPA on Ad:CaPi-mediated genetransfer was also assayed. Control explants infected apically withAdenovirus:CaPi coprecipitates alone had low levels of gene transfer(see FIG. 6D). However, if uPA was first applied to the apical surfaceand followed by the application of the Adenovirus:CaPi coprecipitate,there was an approximately 8-fold increase in gene transfer (see FIGS.6E and 6F). This section of X-gal-stained explants showed both ciliatedand non-ciliated cells expressed the β-galactosidase gene (see FIG. 6G).

Example 6 uPA Enhances Gene Transfer by Specifically Bound Vector toHuman Airway Epithelia

A. Covalent Attachment of u7-peptide to Adenovirus: Bifunctional PEGmolecules were added to virus at room temperature for 60 minutes toallow coupling of PEG to viral surface proteins. See O'Riordan et al.,Human Gene Ther. 10:1349-1358 (1999) (incorporated herein by reference).Unreacted PEG was separated from the PEGylated virus by CsClcentrifugation as described previously and PEGylated virus (Ad2/PEG) wasfinally dialyzed into phosphate-buffered saline (pH 7.0) containing 5%sucrose. u7-peptide and mutated peptides synthesized to include aterminal cysteine with a free sulfhydryl were dissolved in PBScontaining 5% sucrose to a final concentration of 10 mM and added to1.5×10¹² particles of Ad2/PEG at a final concentration of 1 mM. Thepeptide and PEG virus were allowed to couple for 4 hours at roomtemperature. Unreacted peptide was removed from Ad2/PEG/peptide bydialysis into PBS containing 5% sucrose.

B. Specific Coupling of uPA to Vector Enhances Gene Transfer: Theu7-peptide (SEQ ID NO:1) was coupled to the capsid of an adenovirusexpressing β-galactosidase using PEG as described above. As controls,PEG alone and PEG linked to a mutated peptide (SEQ ID NO:3) were alsocoupled to adenovirus. FIG. 7 shows that coupling of the u7-peptide tothe vector via PEG enhanced gene transfer to airway epithelia 10-fold.

1. A method for generating a cAMP-stimulated current in a chloride ionchannel in cystic fibrosis (CF) airway epithelia comprisingadministering to said CF airway epithelia a delivery vehicle constructwhich comprises a cargo portion, said cargo portion comprising a nucleicacid encoding CFTR, and a uPA portion in an amount sufficient to providea cAMP-stimulated current in the chloride ion channel, wherein the uPAportion facilitates the delivery of the cargo portion to the airwayepithelia.
 2. The method of claim 1, wherein said uPA portion of theconstruct is selected from the group consisting of full-length uPA anduPA lacking a PAI-1 binding region.
 3. The method of claim 1, whereinsaid cargo portion is selected from the group consisting of a virus, amodified virus, a viral vector and a non-viral vector.
 4. The method ofclaim 3, wherein said virus is selected from a group consisting of anadenovirus, a herpes virus, a parvovirus, a poxvirus, and a retrovirus.5. The method of claim 4, wherein said parvovirus is adeno-associatedvirus (AAV).
 6. The method of claim 3, wherein said viral vector isselected from a group consisting of an adenovirus vector, a herpes virusvector, a parvovirus vector, a poxvirus vector, and a retrovirus vector.7. The method of claim 6, wherein said viral vector is an adenovirusvector.
 8. The method of claim 3, wherein said viral vector is an AAVvector.